The fetal mouse is a sensitive genotoxicity model that exposes lentiviral-associated mutagenesis resulting in liver oncogenesis

This article is available open access through the publisher’s website at the link below. Copyright @ 2013 The American Society of Gene & Cell Therapy.Genotoxicity models are extremely important to assess retroviral vector biosafety before gene therapy. We have developed an in utero model that demonstrates that hepatocellular carcinoma (HCC) development is restricted to mice receiving nonprimate (np) lentiviral vectors (LV) and does not occur when a primate (p) LV is used regardless of woodchuck post-translation regulatory element (WPRE) mutations to prevent truncated X gene expression. Analysis of 839 npLV and 244 pLV integrations in the liver genomes of vector-treated mice revealed clear differences between vector insertions in gene dense regions and highly expressed genes, suggestive of vector preference for insertion or clonal outgrowth. In npLV-associated clonal tumors, 56% of insertions occurred in oncogenes or genes associated with oncogenesis or tumor suppression and surprisingly, most genes examined (11/12) had reduced expression as compared with control livers and tumors. Two examples of vector-inserted genes were the Park 7 oncogene and Uvrag tumor suppressor gene. Both these genes and their known interactive partners had differential expression profiles. Interactive partners were assigned to networks specific to liver disease and HCC via ingenuity pathway analysis. The fetal mouse model not only exposes the genotoxic potential of vectors intended for gene therapy but can also reveal genes associated with liver oncogenesis.Imperial College London, the Wellcome Trust, and Brunel University

Expression and characterization of the bacterial mechanosensitive channel MscS in Xenopus laevis oocytes

We have successfully expressed and characterized mechanosensitive channel of small conductance (MscS) from Escherichia coli in oocytes of the African clawed frog, Xenopus laevis. MscS expressed in oocytes has the same single-channel conductance and voltage dependence as the channel in its native environment. Two hallmarks of MscS activity, the presence of conducting substates at high potentials and reversible adaptation to a sustained stimulus, are also exhibited by oocyte-expressed MscS. In addition to its ease of use, the oocyte system allows the user to work with relatively large patches, which could be an advantage for the visualization of membrane deformation. Furthermore, MscS can now be compared directly to its eukaryotic homologues or to other mechanosensitive channels that are not easily studied in E. coli

MscS-like mechanosensitive channels in plants and microbes

The challenge of osmotic stress is something all living organisms must face as a result of environmental dynamics. Over the past three decades, innovative research and cooperation across disciplines have irrefutably established that cells utilize mechanically gated ion channels to release osmolytes and prevent cell lysis during hypoosmotic stress. Early electrophysiological analysis of the inner membrane of Escherichia coli identified the presence of three distinct mechanosensitive activities. The subsequent discoveries of the genes responsible for two of these activities, the mechanosensitive channels of large (MscL) and small (MscS) conductance, led to the identification of two diverse families of mechanosensitive channels. The latter of these two families, the MscS family, consists of members from bacteria, archaea, fungi, and plants. Genetic and electrophysiological analysis of these family members has provided insight into how organisms use mechanosensitive channels for osmotic regulation in response to changing environmental and developmental circumstances. Furthermore, determining the crystal structure of E. coli MscS and several homologues in several conformational states has contributed to our understanding of the gating mechanisms of these channels. Here we summarize our current knowledge of MscS homologues from all three domains of life and address their structure, proposed physiological functions, electrophysiological behaviors, and topological diversity

Beitr\ue4ge zur Geschichte der Ichneumoniden

Volume: 16Start Page: 79End Page: 8

Crosslinking of alkylpolysiloxane films on various types of glass surfaces including fused silica using γ-radiation of a⁶⁰cobalt-source. Comparison to crosslinking by thermal peroxid treatment

Crosslinking of alkylpolysiloxane stationary phases, especially in thick film capillary columns, is useful for the homogenous coating of fused silica and pretreated alkaliglass surfaces. The films of the stationary liquid are immobilized against solvent rinsing using CH2Cl2, pentane, and acetone, and maintain homogenity even at high temperature. Various doses of γ-radiation from a60Co. source were used for the crosslinking instead of the thermal peroxid treatment recently described by other authors. The effect of the γ-radiation crosslinking-procedure was investigated in comparison to the peroxid method in regard of: decrease of stationary phase content by solvent rinsing, separation efficiency, tailing behaviour, and bleeding of the columns obtained. Similar results as with the cumylperoxid-treatment were achieved using the γ-radiation-method. By γ-radiation no polar functional groups or moleculs are introduced into the stationary phase, however, as with the peroxid method. Less than 20% of the various stationary liquids are usually removed from the columns by solvent rising after crosslinking using both methods depending on the doses of radiation and the cumylperoxid concentration applied respectively